Deciphering dynamic combinatorial libraries of glycoclusters with miniaturized weak affinity chromatography coupled with mass spectrometry (nano-FAC-MS).

We report an original methodology based on affinity chromatography coupled with mass spectrometry to decipher the complexity of dynamic combinatorial libraries (DCLs) of glycoclusters. Such libraries are intended to boost the design of potential therapeutic anti-infectious agents targeting Pseudomonas aeruginosa, which is responsible for numerous diseases, mostly found in hospitals as major a cause of nosocomial infections. Dynamic combinatorial chemistry provides a rapid access to an equilibrating mixture of glycocluster candidates through the formation of reversible covalent bonds under thermodynamic control. Identifying each molecule in the complex mixture overcomes challenges due to the dynamic process. Selection of glycoclusters candidates was first realized on a model lectin (Concanavalin A, ConA). Home-made affinity nanocolumns, containing covalently immobilized ConA and have volumes in the microliter range, were used to separate DCLs of glycoclusters with respect to their specific lectin binding properties under buffered aqueous conditions. Miniaturization facilitates the inline coupling with MS detection in such purely aqueous and buffered conditions and reduces target protein consumption. Monolithic lectin-affinity columns prepared by immobilization of ConA were first characterized using a known ligand. The amount of active binding immobilized lectin is 61 ± 5 pmol on 8.5-cm length column. We demonstrated the ability of our approach to evaluate individual dissociation constants of species directly in the complex mixture. The concept was then successfully applied to the screening of DCLs of more complex glycoclusters to identify (by mass spectrometry) and rank the ligands (by relative breakthrough curve delay) according to their affinity for the immobilized lectin in a single experiment.

Correction: Fluorescent glycoconjugates and their applications.

Correction for 'Fluorescent glycoconjugates and their applications' by Baptiste Thomas et al., Chem. Soc. Rev., 2020, 49, 593-641, DOI: 10.1039/C8CS00118A.

A general strategy to the intracellular sensing of glycosidases using AIE-based glycoclusters.

Glycosidases, which are the enzymes responsible for the removal of residual monosaccharides from glycoconjugates, are involved in many different biological and pathological events. The ability to detect sensitively the activity and spatiotemporal distribution of glycosidases in cells will provide useful tools for disease diagnosis. However, the currently developed fluorogenic probes for glycosidases are generally based on the glycosylation of the phenol group of a donor-acceptor type fluorogen. This molecular scaffold has potential drawbacks in terms of substrate scope, sensitivity because of aggregation-caused quenching (ACQ), and the inability for long-term cell tracking. Here, we developed glycoclusters characterized by aggregation-induced emission (AIE) properties as a general platform for the sensing of a variety of glycosidases. To overcome the low chemical reactivity associated with phenol glycosylation, here we developed an AIE-based scaffold, which is composed of tetraphenylethylene conjugated with dicyanomethylene-4H-pyran (TPE-DCM) with a red fluorescence emission. Subsequently, a pair of dendritic linkages was introduced to both sides of the fluorophore, to which six copies of monosaccharides (d-glucose, d-galactose or l-fucose) were introduced through azide-alkyne click chemistry. The resulting AIE-active glycoclusters were shown to be capable of (1) fluorogenic sensing of a diverse range of glycosidases including ß-d-galactosidase, ß-d-glucosidase and α-l-fucosidase through the AIE mechanism, (2) fluorescence imaging of the endogenous glycosidase activities in healthy and cancer cells, and during cell senescence, and (3) glycosidase-activated, long-term imaging of cells. The present study provides a general strategy to the functional, in situ imaging of glycosidase activities through the multivalent display of sugar epitopes of interest onto properly designed AIE-active fluorogens.

30 TW and 33 fs pulses delivered by a Ti:Sa amplifier system seeded with a frequency-doubled fiber laser.

We report a full experimental comparison study on the injection of a Ti:Sa multi-terawatt amplifier chain with a standard 15 fs Ti:Sa oscillator and 35 fs frequency-doubled fiber oscillator. The study highlights that the Ti:Sa oscillator, with high performance in terms of pulse duration and spectral width, can be replaced by the frequency-doubled fiber oscillator to seed Ti:Sa amplifier chains almost without any compromise on the output pulse duration and picosecond contrast. Finally, we demonstrate for the first time to our knowledge a 30 TW and 33 fs Ti:Sa amplifier injected by a fiber oscillator.

Supramolecular Assembly of TPE-Based Glycoclusters with Dicyanomethylene-4H-pyran (DM) Fluorescent Probes Improve Their Properties for Peroxynitrite Sensing and Cell Imaging.

Two red-emitting dicyanomethylene-4H-pyran (DM) based fluorescent probes were designed and used for peroxynitrite (ONOO- ) detection. Nevertheless, the aggregation-caused quenching effect diminished the fluorescence and restricted their further applications. To overcome this problem, tetraphenylethylene (TPE) based glycoclusters were used to self-assemble with these DM probes to obtain supramolecular water-soluble glyco-dots. This self-assembly strategy enhanced the fluorescence intensity, leading to an enhanced selectivity and activity of the resulting glyco-dot comparing to DM probes alone in PBS buffer. The glyco-dots also exhibited better results during fluorescence sensing of intracellular ONOO- than the probes alone, thereby offering scope for the development of other similar supramolecular glyco-systems for chemical biological studies.

Multifunctional isoxazolidine derivatives as α-amylase and α-glucosidase inhibitors.

A series of novel isoxazolidines based on benzaldehyde derivatives have been synthesized from the cycloaddition of chiral menthone-based nitrone and allyl phenyl ethers. All synthetic compounds were assessed for their in vitro PPA, HPA and HLAG inhibitory activity. The results revealed that all targets exhibited better inhibitory effect against PPA (12.3 ± 0.4 < IC50 < 38.2 ± 0.9 µM), HPA (10.1 ± 0.4 < IC50 < 26.8 ± 0.2 µM) and HLAG (65.4 ± 1.2 < IC50 < 274.8 ± 1.1 µM) when compared with the reference inhibitor, acarbose (IC50 = 284.6 ± 0.3 µM for PPA, 296.6 ± 0.8 µM for HPA, 780.4 ± 0.3 µM for HLAG) with the highest PPA inhibitory activity was ascribed to compound 3g against both PPA and HPA, and 3b against HLAG enzymes, respectively. Structural activity relationships (SARs) were also established for all synthesized compounds and the interaction modes of the most potent inhibitors (3g for PPA and HPA, 3b for HLAG) and the active site with residues of three enzymes were confirmed through molecular docking studies. Furthermore, a combination of molecular docking analysis with the in vitro activities can help to improve prediction success and encourages the uses of some of these molecules as potential alternatives toward the modulation of T2D.

Glucose-based spiro-oxathiazoles as in vivo anti-hyperglycemic agents through glycogen phosphorylase inhibition.

The design of glycogen phosphorylase (GP) inhibitors targeting the catalytic site of the enzyme is a promising strategy for a better control of hyperglycaemia in the context of type 2 diabetes. Glucopyranosylidene-spiro-heterocycles have been demonstrated as potent GP inhibitors, and more specifically spiro-oxathiazoles. A new synthetic route has now been elaborated through 1,3-dipolar cycloaddition of an aryl nitrile oxide to a glucono-thionolactone affording in one step the spiro-oxathiazole moiety. The thionolactone was obtained from the thermal rearrangement of a thiosulfinate precursor according to Fairbanks' protocols, although with a revisited outcome and also rationalised with DFT calculations. The 2-naphthyl substituted glucose-based spiro-oxathiazole 5h, identified as one of the most potent GP inhibitors (Ki = 160 nM against RMGPb) could be produced on the gram-scale from this strategy. Further evaluation in vitro using rat and human hepatocytes demonstrated that compound 5h is a anti-hyperglycaemic drug candidates performing slightly better than DAB used as a positive control. Investigation in Zucker fa/fa rat model in acute and subchronic assays further confirmed the potency of compound 5h since it lowered blood glucose levels by ∼36% at 30 mg kg-1 and ∼43% at 60 mg kg-1. The present study is one of the few in vivo investigations for glucose-based GP inhibitors and provides data in animal models for such drug candidates.

Fluorescent glycoconjugates and their applications.

Glycoconjugates and their applications as lectin ligands in biology have been thoroughly investigated in the past decades. Meanwhile, the intrinsic properties of such multivalent molecules were limited essentially to their ability to bind to their receptors with high selectivity and/or avidity. The present review will focus on multivalent glycoconjugates displaying an additional capability such as fluorescence properties not only for applications toward imaging of cancer cells and detection of proteins or pathogens but also for drug delivery systems toward targeted cancer therapy. This review is a collection of research articles discussed in the context of the structural features of fluorescent glycoconjugates organized according to their fluorescent core scaffold and with their representative applications.

Lectin PLL3, a Novel Monomeric Member of the Seven-Bladed ß-Propeller Lectin Family.

The Photorhabdus species is a Gram-negative bacteria of the family Morganellaceae that is known for its mutualistic relationship with Heterorhabditis nematodes and pathogenicity toward insects. This study is focused on the characterization of the recombinant lectin PLL3 with an origin in P. laumondii subsp. laumondii. PLL3 belongs to the PLL family of lectins with a seven-bladed ß-propeller fold. The binding properties of PLL3 were tested by hemagglutination assay, glycan array, isothermal titration calorimetry, and surface plasmon resonance, and its structure was determined by X-ray crystallography. Obtained data revealed that PLL3 binds similar carbohydrates to those that the other PLL family members bind, with some differences in the binding properties. PLL3 exhibited the highest affinity toward l-fucose and its derivatives but was also able to interact with O-methylated glycans and other ligands. Unlike the other members of this family, PLL3 was discovered to be a monomer, which might correspond to a weaker avidity effect compared to homologous lectins. Based on the similarity to the related lectins and their proposed biological function, PLL3 might accompany them during the interaction of P. laumondii with both the nematode partner and the insect host.

Thiophenol detection using an AIE fluorescent probe through self-assembly with TPE-based glycoclusters.

We describe a novel green-emitting tetraphenylethylene-dicyanomethylene-4H-pyran (TPE-DCM) based fluorescent probe (TD-1). Conjugating TPE and DCM moieties allowed TD-1 to display high selectivity for thiophenol with excellent AIE properties in aqueous solution. Nevertheless, the poor water solubility of the hydrophobic structure resulted in a weak and unstable emission intensity. The non-covalent self-assembly of TD-1 with a TPE glycocluster (TPE2S) led to a largely improved water solubility producing a reliable and stable sensing system. The corresponding glyco-probe could sensitively detect exogenous thiophenol concentrations in PBS buffer or environmental water samples.

Deciphering multivalent glycocluster-lectin interactions through AFM characterization of the self-assembled nanostructures.

Pseudomonas aeruginosa is a human opportunistic pathogen responsible for lung infections in cystic fibrosis patients. The emergence of resistant strains and its ability to form a biofilm seem to give a selective advantage to the bacterium and thus new therapeutic approaches are needed. To infect the lung, the bacterium uses several virulence factors, like LecA lectins. These proteins are involved in bacterial adhesion due to their specific interaction with carbohydrates of the host epithelial cells. The tetrameric LecA lectin specifically binds galactose residues. A new therapeutic approach is based on the development of highly affine synthetic glycoclusters able to selectively link with LecA to interfere with the natural carbohydrate-LecA interaction. In this study, we combined atomic force microscopy imaging and molecular dynamics simulations to visualize and understand the arrangements formed by LecA and five different glycoclusters. Our glycoclusters are small scaffolds characterized by a core and four branches, which terminate in a galactose residue. Depending on the nature of the core and the branches, the glycocluster-lectin interaction can be modulated and the affinity increased. We show that glycocluster-LecA arrangements highly depend on the glycocluster architecture: the core influences the rigidity of the geometry and the directionality of the branches, whereas the nature of the branch determines the compactness of the structure and the ease of binding.

Tetraphenylethylene-based glycoclusters with aggregation-induced emission (AIE) properties as high-affinity ligands of bacterial lectins.

Tetraphenylethylene (TPE) is fluorescent through aggregation induced emission (AIE) in water. Herein, TPE was used as the core of glycoclusters that target the bacterial lectins LecA and LecB of Pseudomonas aeruginosa. Synthesis of these TPE-based glycoclusters was accomplished by using azide-alkyne "click" chemistry. The AIE properties of the resulting glycoclusters could be readily verified, but imaging could not be pursued due to the overlap of the fluorescence signals from cells and bacteria. Nonetheless, the glycoclusters displayed nanomolar affinities toward LecA and LecB. Further evaluation in a cell-based anti-adhesive assay highlighted a limited decrease in adhesion (20%) for the fucosylated glycocluster. This confirmed that these TPE-based glycoclusters are indeed LecA and LecB high-affinity ligands. Nevertheless, the hypotheses involving their application in imaging or anti-adhesive therapy could not be verified.

The anti-adhesive effect of glycoclusters on Pseudomonas aeruginosa bacteria adhesion to epithelial cells studied by AFM single cell force spectroscopy.

The human opportunistic pathogen Pseudomonas aeruginosa (PA) is responsible for chronic infections of the respiratory epithelium in cystic fibrosis patients. PA takes advantage of an arsenal of virulence factors to infect and colonize human lungs. Among them, the lectin LecA favours epithelium invasion by interacting with host cell globotriaosylceramide (Gb3). A new therapeutic approach is based on the development of synthetic multivalent molecules (glycoclusters) targeting LecA with a higher affinity than its natural ligand. Atomic force microscopy-single cell force spectroscopy has been used to study the effect of glycoclusters on the bacteria-cell interaction. Glycoclusters have been shown to affect the detachment work and detachment force of the bacteria-cell interaction. The specificity and the efficiency of the glycocluster in targeting the lectin and destabilizing the PA-epithelial cell adhesion are demonstrated and discussed.

Fixing the Conformation of Calix[4]arenes: When Are Three Carbons Not Enough?

Calix[4]arenes are unique macrocycles that through judicious functionalisation at the lower rim can be either fixed in one of four conformations or remain conformationally flexible. Introduction of propynyl or propenyl groups unexpectedly provides a new possibility; a unidirectional conformational switch, with the 1,3-alternate and 1,2-alternate conformers switching to the partial cone conformation, whilst the cone conformation is unchanged, under standard experimental conditions. Using 1 H NMR kinetic studies, rates of switching have been shown to be dependent on the starting conformation, upper-rim substituent, where reduction in bulk enables faster switching, solvent and temperature with 1,2-alternate conformations switching fastest. Ab initio calculations (DFT) confirmed the relative stabilities of the conformations and point towards the partial cone conformer being the most stable of the four. The potential impact on synthesis through the "click" reaction has been investigated and found not to be significant.

Perylenediimide-based glycoclusters as high affinity ligands of bacterial lectins: synthesis, binding studies and anti-adhesive properties.

The synthesis of eight perylenediimide-based glycoclusters was readily performed from hexa- and tetra-propargylated cores through azide-alkyne "click" conjugation. Variations in the carbohydrate epitope (Glc, Gal, Man, Fuc) and the linker arm provided molecular diversity. Interactions with LecA and LecB, two proteins involved in the adhesion of Pseudomonas aeruginosa to host tissues, were evaluated by microcalorimetry (ITC). In both cases high affinities were obtained with Kd values in the nanomolar range. Further evaluation of their anti-adhesive properties using cultured epithelial cells demonstrated their potent anti-adhesive activities against Pseudomonas aeruginosa with only 30-40% residual adhesion observed. The fluorescence properties of the PDI core were then investigated by confocal microscopy on cell-bacteria cultures. However, the red fluorescence signal of the PDI-based glycocluster was too weak to provide significant data. The present study provides another type of anti-adhesive glycocluster against bacterial infection with a large aromatic PDI core.

Supramolecular assembly of fluorogenic glyco-dots from perylenediimide-based glycoclusters for targeted imaging of cancer cells.

Supramolecular self-assembly between perylenediimide-based glycoclusters and a red-emitting fluorophore produces structurally uniform and stable glyco-dots amenable to targeted fluorogenic imaging of liver and triple-negative breast cancer cells.

Naphthyl Thio- and Carba-xylopyranosides for Exploration of the Active Site of ß-1,4-Galactosyltransferase 7 (ß4GalT7).

Xyloside analogues with substitution of the endocyclic oxygen atom by sulfur or carbon were investigated as substrates for ß-1,4-galactosyltransferase 7 (ß4GalT7), a key enzyme in the biosynthesis of glycosaminoglycan chains. The analogues with an endocyclic sulfur atom proved to be excellent substrates for ß4GalT7, and were galactosylated approximately fifteen times more efficiently than the corresponding xyloside. The 5a-carba-ß-xylopyranoside in the d-configuration proved to be a good substrate for ß4GalT7, whereas the enantiomer in the l-configuration showed no activity. Further investigations by X-ray crystallography, NMR spectroscopy, and molecular modeling provided a rationale for the pronounced activity of the sulfur analogues. Favorable π-π interactions between the 2-naphthyl moiety and a tyrosine side chain of the enzyme were observed for the thio analogues, which open up for the design of efficient GAG primers and inhibitors.

Design and Synthesis of Galactosylated Bifurcated Ligands with Nanomolar Affinity for Lectin LecA from Pseudomonas aeruginosa.

Lectin A (LecA) from Pseudomonas aeruginosa is an established virulence factor. Glycoclusters that target LecA and are able to compete with human glycoconjugates present on epithelial cells are promising candidates to treat P. aeruginosa infection. A family of 32 glycodendrimers of generation 0 and 1 based on a bifurcated bis-galactoside motif have been designed to interact with LecA. The influences both of the central multivalent core and of the aglycon of these glycodendrimers on their affinity toward LecA have been evaluated by use of a microarray technique, both qualitatively for rapid screening of the binding properties and also quantitatively (Kd ). This has led to high-affinity LecA ligands with Kd values in the low nanomolar range (Kd =22 nm for the best one).

C-Glycopyranosyl Arenes and Hetarenes: Synthetic Methods and Bioactivity Focused on Antidiabetic Potential.

This Review summarizes close to 500 primary publications and surveys published since 2000 about the syntheses and diverse bioactivities of C-glycopyranosyl (het)arenes. A classification of the preparative routes to these synthetic targets according to methodologies and compound categories is provided. Several of these compounds, regardless of their natural or synthetic origin, display antidiabetic properties due to enzyme inhibition (glycogen phosphorylase, protein tyrosine phosphatase 1B) or by inhibiting renal sodium-dependent glucose cotransporter 2 (SGLT2). The latter class of synthetic inhibitors, very recently approved as antihyperglycemic drugs, opens new perspectives in the pharmacological treatment of type 2 diabetes. Various compounds with the C-glycopyranosyl (het)arene motif were subjected to biological studies displaying among others antioxidant, antiviral, antibiotic, antiadhesive, cytotoxic, and glycoenzyme inhibitory effects.